Tobias Riedl, Department: Chronic Inflammation and Cancer (F180), Deutsches Krebsforschungszentrum (DKFZ)
Amir Keric, OMNI Life Science GmbH & Co. KG
Mathias Heikenwälder, Department: Chronic Inflammation and Cancer (F180), Deutsches Krebsforschungszentrum (DKFZ)
Collectively, this study represents the first effort to differentiate the HepaRG, HepG2 and Huh7 cell lines as 3D spheroids in the novel 3D automated culture system “CERO”. To support current research it becomes particularly important to enhance and standardize culture conditions of abovementioned cell lines.
Currently, the ultra-low attachment plates are mostly used for spheroid generation and differentiation, although this technique still holds limitations such as labour intensity, susceptibility to contaminations, spatial and time limitations. Moreover, spheroids of different hepatic cell lines become necrotic and subsequently fall apart, starting one week after seeding. Taking into account that the establishment of a self-sustaining HBV infection takes roughly 7 to 10 days, this method is not suitable.
Consequently, to overcome those limitations, new approaches have been developed. In this project, the feasibility of the CERO was evaluated with respect to cell proliferation and differentiation as 3D spheroids. Here it can be shown, that large numbers of spheroids originating from the same batch of cells, were kept viable for a long-term cell culture period, e.g. HepaRG 80 days, Huh7 20 days and HepG2 >40 days. These spheroids were stained positive for carbonic anhydrase IX (CAIX), which is widely accepted and used as a marker for hypoxia. In addition to enhanced viral replication of hepatitis B virus (HBV) and hepatitis C virus (HCV) under hypoxic conditions1,2, we could show that the cell intrinsic anti-HBV factor apolipoprotein B mRNA editing enzyme catalytic subunit 3B is down-regulated under hypoxic conditions3.
Our observations from sections of human liver biopsies of chronic HBV (CHB) patients lead to the conclusion, that viral replication is enhanced in peripheral regions of the liver, going hand in hand with ak.k decrease of APOBEC3B mRNA4. It could be shown, that Huh7 spheroids, generated and differentiated for 10 days in the CERO are susceptible to HCV infection, as well as HepG2-NTCP spheroids, generated in the same way and differentiated for 40 days are susceptible for HBV infection.
This rises the hope that “CERO” can be efficiently used to generate spheroids featuring oxygen gradients similar to those seen in the liver on a smaller scale; and study replication of hepatotropic viruses along those, which was not easy, if, at all possible.